ABSTRACT

Antigonon leptopus flower extract was tested for its anticoagulant effect on human blood. Five hundred (500.00) grams of A.leptopus flowers were macerated using a blender and the juice were squeezed to obtain the extract. Control and experimental set-ups with three replications each were prepared. Each replication in the control set-up was prepared by mixing 2.00 mL of distilled water with 2.00 mL of fresh blood in a vacuum tube while each replication in the experimental set-up was prepared by mixing 2.00 mL of the prepared extract with 2.00 mL of fresh blood in a vacuum tube. Microscopic observation and direct vacuum tube observations were done to note the time of blood to visibly coagulate in each trial in each set-up. In the microscopic observation the experimental and control set-ups had an average time elapsed for the presence of blood coagulation of 90.67 and 26.67 minutes respectively. The experimental and control set-ups had an average time elapsed for blood clots to be visible of 38.34 and 0.83 hours respectively in the vacuum tube observation. Two-way ANOVA at 0.01 level of significance, degrees of freedom: (2,5) for treatment showed that there is a significant difference between the set-ups in both microscopic and vacuum tube observation in the time elapsed for blood coagulation to be visible. Based on the results, the A.leptopus flower extract can be used as an effective anticoagulant on human blood.

Science Investigatory Project presented in partial fulfillment of the requirements for graduation of the Revised Basic Education Curriculum (RBEC) to the panelists and concerned staff of Bantayan Science High School, Bantayan, Cebu. Prepared under the direction and guidance of Mr. Ulysis Tisado.

INTRODUCTION Nature, Importance and Rationale of the Study The shelf-life of human blood is just a short period of time due to the rapid coagulation of the said body fluid. Certain medical purposes need a longer period of time for the usage of blood (Cabatit, 1985). So, it is necessary to lengthen the shelf-life of blood by delaying its coagulation or in other words applying a blood anticoagulant for longer storage and time of usefulness. Anticoagulant decreases the normal range of bloods coagulation time; hence, blood can be stored at a longer time maintaining its physical state and fluidity. Because of this, a study, which concerns about the possibility of delaying the coagulation time of blood by making blood anticoagulants from plant parts specifically A. leptopus flowers, was conducted. A.leptopus locally known as Cadena de Amor is a climbing, perennial vine. Its stems attain a length of 10 meters and leaves are alternate up to 10 cm long. Flowers are white or pale to deep pink up to 2 cm long, with a 5-parted and persisted perianth. Fruit is an ovoid achene and is about 1 cm long and broad at the base, narrowing towards the tip, 1

and is loosely surrounded by the persistent lobes of the flower. Problem and Objectives of the Study The study aimed to answer the following problems: 1. What is the effect of A.leptopus flower extract to the coagulation time of blood as compared to that of the distilled water? 2. Is A.leptopus flower extract a possible blood anticoagulant? The study aimed to test if A.leptopus flower extract has a blood anticoagulant effect. It target delaying the time of blood coagulation and lengthening the shelf-life of blood samples. It focused on evaluating the effect of A.leptopus flower extract on the coagulation time of blood as compared to that of the distilled water.

Limitations of the Study The study was limited only to the extraction of A. leptopus flower, the possibility of the said extract as blood anticoagulant and the quantitative comparison of coagulation time of blood under both microscopic and vacuum-tube observation of the extract and distilled water. Chemical tests and analysis of the extract was not conducted. A two-way ANOVA was employed for the time elapsed for blood to coagulate to be visible under both vacuumtube and microscopic observations.

REVIEW OF RELATED LITERATURE Coagulation Coagulation is a complex process by which blood forms clots. It involves a cellular (platelet) and proteins (coagulation factors) (http://medicaldictionary.thefreedictionary.com/Coagulation+Disorders). Blood coagulation is the process of transformation of a liquid into a soft, semisolid, or a solid mass. Blood coagulation is characterized by thrombosis-the formation, presence, or development of a thrombus, a blood clot formed (Cabatit, 1985). Coagulation Time The coagulation time is the time by which the blood takes a clot. Normally in an ordinary skin puncture, blood coagulation takes place in 2-6 minutes. It is affected by such factors as temperature, size of the drop, and smoothness and cleanliness of the instrument. But the normal coagulation time when blood is taken from a vein may reach twenty minutes. (Cabatit, 1985) Cadena de Amor Chain of love, Antigonon leptopus, locally known as Cadena de Amor is a climbing, perennial vine. Its stems attain a length of 10 meters and leaves are alternate up to 10 cm long. Flowers are white or pale to deep pink up to 2 cm long, with a 5-parted and persisted perianth. Fruit is an ovoid achene and is about 1 cm long and broad at the base, narrowing towards the tip, and is loosely surrounded by the persistent lobes of the flower. Studies have shown that it has anti-thrombin, analgesic, anti-inflammatory, ant-diabetic and lipid peroxidation inhibitory properties. The anti-thrombin property was observed under a methanol extract of the plant. A.leptopus is found to be edible and practiced by some as medical remedies for certain ailments and diseases. In some parts of the world, the tubers and flowers are consumed as food; on the other hand, in Thailand, leaves are fried, coated with flour and served with noodles. Ifugao in the Philippines, use Cadena de amor for wound closure. In 3

Trinidad and Tobago, the plant is used for diabetes, low blood pressure, and as a heart tonic. In Jamaica, decoction of flowers and aerial parts are used for cold remedies. (http://stuartxchange.com/CadenaDeAmor.html) Coagulation Components There are many factors and substances that make up the coagulation process namely platelets, platelet aggregators, and fibrin-formed from fibrinogen. The coagulation system is comprised by coagulation factors: Factor XII, Factor XI, Factor X, Factor VII and Factor V. On the other hand, coagulation is prevented and delayed by application of blood anticoagulants, vasoconstrictors, and platelet aggregator inhibitors. Blood anticoagulants include heparin, coumadin and many more. (http://www.naturdoctor.com/Chapters/Research/thrombosis_prevention.html) Coagulation as a Complex Process The sequence of events involving the process of coagulation (after tissue injury and platelet destruction) are: 1. Platelet factors + Plasma factors Plasma thromboplastin (AHG, PTC, PTA) 2. Plasma thromboplastin + (LF, SF, Ca) thromboplastin complex 3. Thromboplastin complex + Prothrombin thrombin Platetelet acc. 1 4. Thrombin + fibrinogen Fibrin (insoluble clot) Platelet acc.2 In the conversion of fibrinogen to fibrin, the 3 steps are: 1. proteolysis of fibrinogen 2. soft clot formation 3. hard clot formation The clot formation is affected by the pH because the fibrin monomer aggregation of the soft clot formation is affected by the strength of the hydrogen bonding and pH. 4

(Cabatit, 1985) Blood anticoagulants Here are some inhibitors of blood coagulation or anticoagulants: 1. Heparin- a complex carbohydrate and is believed to be formed by the mast cells and rapidly destroyed by heparinase. It inhibits activatiuon of prothrombin and inactivates thrombin. 2. Antithrombin- present in small amount in plasma and inactivates thrombin. 3. Antithromboplastin- a lipid that inactivates thromboplastin. 4. Dicumarol- prevents the body from utilizing vitamin K: thus, it depresses the production of proconvertin which is needed for blood coagulation. 5. Fibrinolytic substances- present in blood and tissues of normal persons. It limits intravascular clotting. Water as Amphoteric At pH 7 water is neutral.But...water is amphoteric- it can act as an acid or a base if the pH of 7 is disrupted. Its acidic or base like properties depend on whether it is receiving or donating a proton. (acids-proton donors, bases-proton acceptors). (http://wiki.answers.com/Q/Is_water_a_base_or_an_acid) Statistical Parameter A two-way Analysis of Variance is a statistical parameter to determine if there is a significant difference between the existing treatments and replications. It is used if 2 or more variables are involved.

METHODOLOGY Materials: Blender pH paper wire gauze 125.00 mL Erlenmeyer flask 106.00 mL Distilled water Dropper 12.00 mL Fresh blood samples 12 vaccuum tubes 3 microscopes GENERAL PROCEDURE: *Collection of flowers Pink-colored flowers of A.leptopus were collected and weighed. Before weighing the fruits were separated from the flowers. *Extraction A. leptopus flowers Five hundred (500.00) grams of collected A.leptopus flowers were macerated with 100.00 mL of distilled water using a blender. The macerated flowers were decanted to a cheese cloth and were squeezed to obtain the extract. The extract was transferred into an eflask. The pH, volume, color and odor of the extract were noted. *Cleaning of vacuum tubes The vacuum tubes were sterilized by subjecting them to boiling water placed in a beaker. 6 slides and cover slips 250.00 mL beaker alcohol lamp iron rings iron stands 100.00 mL graduated cylinder Cheese cloth Triple beam balance 500.00 g A.leptopus flowers

*Preparation of the Reagents Three vacuum tubes were filled with 2 mL each of the prepared extract. This composed the experimental set-up. Another three vacuum tubes were filled with 2 mL each of distilled water, which composed the control set-up. *Collection of Blood Samples Blood was freshly extracted from two persons, labeled as A and B, by the Medical Technician of Bantayan District Hospital. Six vacuum tubes were filled by 2 mL each of the freshly extracted blood. *Preparation of the Set-ups Right after the blood was decanted into the vacuum tubes, they were immediately mixed with the prepared reagents namely the A. leptopus flower extract and the distilled water. The following set-ups were made: Set-ups Experimental Trial 1 2 mL blood(A) + 2 mL A. leptopus Control Trial 2 Trial 3 2 mL blood(B) + 2 2 mL blood(A) + 2 mL A. leptopus mL A. leptopus

*A and B are the source of the blood *Microscopic Observation The set-ups were continuously observed under the microscope (400x) by getting a small amount of each sample using a dropper and putting them into the slides. After the slide was used it was subjected to boiling water for sterilization for future use. Supposedly, the observation interval must be one minute, but, considering that getting the specimens, putting them into the slides, and focusing them clearly under the microscope takes more than two minutes, a fixed interval was already neglected. Instead of fixing the interval, an immediate continuous microscopic observation was made. It means that after 7

a few minutes of observing under the microscope for determining the presence of blood coagulation, new samples were immediately obtained and subjected to the said observation. One researcher was responsible for taking the notes of every microscopic observation finished and was the one taking the exact time for the said confirmation of the presence of blood coagulation as viewed microscopically for every trial in each set-up. Each time the blood coagulation was present and observed in a certain blood sample, further microscopic observation to that certain sample was already being stopped. *Vacuum tube observation Simultaneous to the microscopic observation, a vacuum-tube observation was also conducted by viewing from the image formed as being viewed at the outside of the vacuum-tube if it had visibly coagulated already. The time and date for insoluble blood clot to be visible under vacuum tube observation for each of the trials of every set-up was noted. *Data Processing The data obtained on both microscopic and vacuum tube observation cannot be used as a statistical data; hence, the obtained data were processed for presentation of results and statistical use. The time (in minutes) elapsed for the presence of blood coagulation under microscopic observation was obtained by using the equation below. (HHMM)f where: (HHMM)f (HHMM)i = HH x

- noted time for presence of blood coagulation

To obtain the time elapsed, we subtract the noted time for the presence of blood

coagulation by the time for the blood sample to be obtained. The equation neglected the date and time position (AM/ PM) because the data obtained had the same date, February 3, 2012, and time position, PM. The elapsed hour time was converted into minutes using a conversion factor and then added with the elapsed minute time to obtain the time elapsed in minutes for the presence of blood coagulation under microscopic observation. The time (in hours) elapsed for insoluble blood clots to be visible was obtained by using the equation below. [ (DDf- DDt) x X + (HHMM)f] - (HHMM)i = HH + MM x where: (HHMM)f (HHMM)i HH MM DDf DDt - noted time for presence of blood coagulation - time for blood sample to be freshly obtained - hour time in 24- hour time format - minute time - noted date for insoluble blood clot to be visible - date for blood sample to be freshly obtained - 12 if (HHMM)f is less than (HHMM)i - 24 if (HHMM)f greater than (HHMM)i [ (DDf- DDt) x X + (HHMM)f] modified noted time for insoluble blood clot to 1 be visible HH + MM x

1 60

60)) elapsed in hours - time

To obtain the time elapsed, first, the date for blood sample to be freshly obtained subtracted from the noted date for insoluble blood clot to be visible. And then, multiplied the obtained value with the corresponding hour equivalent and added it to the noted time for insoluble blood clots to be visible. The obtained value was already the modified noted time. We subtract the modified noted time for insoluble blood clots to be visible by the time for the blood sample to be obtained. The elapsed minute time was converted into hours using a conversion factor and then added with the elapsed hour time to obtain the time elapsed in hours for insoluble blood clot to be visible under vacuum tube observation. 9 *Incineration of Blood Samples

After the blood samples were already observed by all the data and information needed, they were incinerated and the vacuum tubes were disposed for safety and sanitary purposes. *Statistical Parameter and Analysis Since the statistical parameter used is two-way ANOVA, we need to define our hypotheses for replication and treatment to interpret our results in the given parameter. The hypotheses for replication are the following: Null hypothesis: There is no significant difference between the time (in minutes) elapsed for blood to coagulate under microscopic observation or time (in hours) elapsed for insoluble blood clots to be visible under vacuum tube observation in every replication or trial; therefore, Ho is R1 = R2 = R3 where: R- replication or trial Alternative hypothesis: There is a significant difference between the time (in minutes) elapsed for blood to coagulate under microscopic observation or time (in hours) elapsed for insoluble blood clots to be visible under vacuum tube observation in every replication or trial; therefore, Hi is R1 R2 R3 where: R- replication or trial The hypotheses for treatment are the following: Null hypothesis: There is no significant difference between the time (in minutes) elapsed for blood to coagulate under microscopic observation or time (in hours) elapsed for insoluble blood clots to be visible under vacuum tube observation in every treatment- blood with A. leptopus extract and blood with distilled water; therefore, Ho is T1 = T2 where: T- treatment Alternative hypothesis: There is a significant difference between the time (in minutes) elapsed for blood to coagulate under microscopic observation or time (in hours) elapsed for insoluble blood clots to be visible under vacuum tube observation in every treatment- blood with A. leptopus extract and blood with distilled water; therefore, 10 Hi is T1 T2 where: T- treatment

If the obtained F value is greater than or equal to the tabular value at 0.01 level of significance, degrees of freedom: (2,5) for treatment and (1,5) for replication, null hypothesis(Ho) was rejected and alternative hypothesis(Hi) was accepted. The results of this two-way ANOVA are favorable to the study if there is no significant difference between the existing replications. And if there is a significant difference between the treatments, since the comparison is between the A. leptopus flower extract- the tested variable as blood anticoagulant- with the distilled water- an unconsidered anticoagulant.

The A. leptopus flower extract had a pale-brown color, strong, woody odor, and a pH of 4. The obtained extract from the macerated flowers with 100.00 mL distilled water was 70 mL. The extract was acidic based on its pH value. The acidic property of the extract may possibly be caused by the organic materials and other substances contained by the A. leptopus flowers. Those organic materials and substances were obtained by the extract upon squeezing the macerated flowers. The low pH value or the acidic nature may possibly affected the time of clot formation or blood coagulation as it is cited on the Review of Related Literature (see Review; Coagulation as a Complex Process). The extracted volume, 70 mL, was less than that of the volume of the added water, 100 mL, may be attributed by: a. the minimal amount of water which remained in the flowers in 12 the blender during decantation ( as it is observed that the A. leptopus flowers absorbed water); b. the existing amount of water absorbed by the flowers in the residue after the

squeezing ( as it is considered that squeezing is not enough to extract the whole amount of water that is contained by the macerated flowers).

Microscopic Observation The presence of blood coagulation can be observed microscopically by viewing if there is already a presence of cells that have already colonized or grouped or clumped, and if viewed at technically high microscopes, there is already the presence of a clear thrombus. The time by which this presence of coagulation occurs is important because it will lead to the determination of the coagulation time of a certain blood sample. Table 2.1 below shows the elapsed time of blood for its coagulation as it was viewed microscopically. Table 2.1. Time (in minutes) elapsed for the presence of blood coagulation under microscopic observation Set-up Trial 1 Trial 2 Trial 3 Average Experimental 98 88 86 90.67 Control 36 18 26 26.67 Table 2.1 shows the time difference of the time which the blood samples was observed to have the presence of blood coagulation from the time by which the blood sample was freshly obtained and was mixed by the reagents. The control set-up trial 2, obtained the earliest time which is 18 minutes for blood coagulation to be observed, 13 followed by control set-up trial 3 which coagulated 26 minutes after it was obtained, then, followed by control set-up trial 1 which had a time elapsed of 36 minutes. The experimental set-up trial 3 coagulated in the 86th minute, and then experimental set-up trial 2 coagulated after 88 minutes and lastly experimental set-up trial 1 was observed to have the presence of blood coagulation 98 minutes after the blood was freshly obtained.

The coagulation time of the blood in control set-up trials 1 and 3, which were 36 and 26 respectively, were more than the normal time of coagulation, which was 20 minutes, as presented in the review. This few minutes of delay might be caused by the presence of distilled water, which may sometimes act as an acid and sometimes as a base. The nature of the distilled water of its basicity and acidity may also have effected the coagulation time of blood. ( see Review; Water as Amphoteric).

*Statistical Analysis The two way Analysis of Variance (ANOVA) is essential to determine whether or not there is a significant difference between the tested variables. We are also able to determine if there is a significant difference between the replications as presented on the data utilized. This two-way ANOVA will be able to lead us to an interpretation if the A. leptopus flower extract can be used as a blood anticoagulant. Table 2.2 shows the two-way ANOVA of the time elapsed for the presence of blood coagulation under the microscope.

As stated above in the table, there are three sources of variance: the replication, treatment and error. The degrees of freedom of replication and treatment, 2 and 1 respectively, were obtained from the total number of replication of treatments decreased by 1. The degrees of freedom of error, 4, was from the sum of the total number of replications and treatments decreased by 1. The sum of squares for replication, treatment, error were 217.33, 6144, and 28 respectively. The mean squares for replication, treatment, error were 108.87, 6144, and 7 respectively obtained by dividing the sum of squares by their respective degrees of freedom. The F value for replication and treatment, 15.52 and 877.71 respectively, were obtained by dividing each mean square by the mean square of error. The obtained F value for replication, 15.52, is less than the critical value which is 18.00 at 0.01 level of significance; therefore, the Ho is accepted which states that there is no significant difference between the time (in minutes) elapsed for the presence of blood coagulation under microscopic observation in the three trials or replications. It can then be interpreted that the 3 trials or replications has obtained 15 acceptable data from the accepted Ho that the trials have insignificant differences with each other; hence the three trials had a just and fair data based from the statistical result. The F value for treatment, 877.71, is greater than the critical value, 21.20, at 0.01 level of significance. So, there is a significant difference between the the time (in minutes) elapsed for the presence of blood coagulation under microscopic observation of the experimental set-up, the blood with A. leptopus flower extract, and the control set-up, the blood with distilled water. It can then be concluded that the A. leptopus flower extract has

an effect on the delay in the the time (in minutes) elapsed for the presence of blood coagulation under microscopic observation.

Vacuum tube Observation The naked eye observes and detects the presence of blood clot as the blood sample is seen on a transparent container like a vacuum tube. The time of the presence the blood clot had been observed will identify the time by which the blood sample produces an observable, insoluble blood clot, which is a product of blood coagulation. Table 3.1 shows the time (in hours) elapsed for insoluble blood clots to be observed as observed from the outside of the vacuum tube.

Table 3.1 shows the time difference of the time which the blood samples was observed to have the presence of blood coagulation from the time by which the blood sample was freshly obtained and was mixed by the reagents. The control set-up trials 2 and

3 obtained the earliest time elapsed which is 0.82 hours for blood clots to be observed. The remaining trial, trial 3, of the control set-up obtained the next time difference with 0.85 hours. The trials of the experimental set-up garnered the longest time that elapsed. Trial 3 of the said set-up had a 16.7 time elapsed in hours. Next with 49.15 hours of elapsed time, trial 2 was observed with blood clots. And lastly, trial 1 had the longest time difference with 49.18 hours of elapsed time for insoluble blood clots to be observed. The very early coagulation of trial 3 of the experimental set-up as compared to the other trials in the same set-up may possibly be caused by the following events which occurred during the experiment: In taking samples for the blood to be used for microscopic observation, a dropper was just utilized. Because the instrument is not that appropriate for taking blood specimen, spills, decantation errors and other wasteful scenarios had occurred. So, after 17 the microscopic observation was already done, trial 3 was observed to have the least volume remaining in the tube as compared with the other trials. Since the extract was less dense than the blood (as it is observed that the extract floats over the blood in the mixing process), it was more prone to waste and spills. Since the A. leptopus extract is one of the independent variables, which causes the change in the dependent variable- the time for blood clots to be visible under vacuum tube observation- it is possible that the less volume of the extract that remained in the trial caused the early delay of blood clots to be visible as compared to that of the other trials in the experimental set-up.

* Statistical Analysis

The two way Analysis of Variance (ANOVA) is essential to determine whether or not there is a significant difference between the tested variables. We are also able to determine if there is a significant difference between the replications as presented on the data utilized. This two-way ANOVA will be able to lead us to an interpretation if the blood with A. leptopus extract can delay the time elapsed for blood clots to be visible. Table 3.2 shows the two-way ANOVA of the time elapsed for the presence of blood coagulation as it is observed in the vacuum tube.

As stated above in the table, there are three sources of variance: the replication, treatment and error. The degrees of freedom of replication and treatment, 2 and 1 respectively, were obtained from the total number of replication of treatments decreased by 1. The degrees of freedom of error, 4, was from the sum of the total number of replications

and treatments decreased by 1. The sums of squares for replication, treatment, and error were 351.65, 2110.87, and 351 respectively. The mean squares for replication, treatment, and error were 108.87, 6144, and 7 respectively obtained by dividing the sum of squares by their respective degrees of freedom. The F value for replication and treatment, 2.00 and 24.06 respectively, were obtained by dividing each mean square by the mean square of error. The obtained F value for replication, 2.00, is less than the critical value which is 18.00 at 0.01 level of significance; therefore, the Ho is accepted which states that there is no significant difference between the time (in hours) elapsed for blood to coagulate under vacuum tube observation in the three trials or replications. It can 19 then be interpreted that the 3 trials or replications had obtained acceptable data from the accepted Ho that the trials have insignificant differences with each other; hence the three trials had a just and fair data based from the statistical results. The F value for treatment, 877.71, is greater than the critical value, 21.20, at 0.01 level of significance. So, there is a significant difference between the the time (in hours) elapsed for blood to coagulate under vacuum tube observation of the experimental set-up, the blood with A. leptopus flower extract, and the control set-up, the blood with distilled water. It can then be concluded that the A. leptopus flower extract has an effect on the delay in the time (in hours) elapsed for blood to coagulate under vacuum tube observation..

20 CONCLUSIONS AND RECOMMENDATIONS

CONCLUSIONS

Based from the interpretations of the statistical parameter on both time elapsed for visible blood coagulation under microscopic and vacuum tube observations, there is a delay in the coagulation perion of blood with A. leptopus flower extract as compared to that of the distilled water. So, by delaying the coagulation time , it will lengthen the shelf-life of blood. In summary, Cadena de Amor (Antigonon leptopus) flower extract can be used as a blood anticoagulant.

RECOMMENDATIONS

Conduct a chemical test on the A. leptopus flower extract to know what component of the extract causes the anticoagulant activity on human blood

Compare the effectiveness of the extract as blood anticoagulant with existing and commonly used blood anticoagulants

Let the treated blood undergo in a centrifuge machine to clearly view the size and presence of the insoluble blood clots

Utilize a more appropriate device or instrument for taking the specimen to be used in microscopic observation for better accuracy and fairness to each trial

21 Conduct a study if the A. leptopus flower extract has an effect to the physical and chemical characteristics and composition of blood other than coagulation time which may affect the needed blood components for medical and other purposes Use a blood sample from one person only throughout the experiment for a more just experiment Utilize a more highly powered and technically high microscopes for easy and fast viewing of blood coagulation; in this case, the microscopic determination would be easier and faster; thus, it would take a little time and a fixed small time interval can be utilized in the microscopic observation, which produces a more accurate result Conduct another study which focuses on the effectiveness of A. leptopus flower extract with respect to the ratio of the volume of the extract and the volume of the blood sample treated.